• Identification of Anti-Beta2 Glycoprotein I Auto-antibody Regulated Gene Targets in the Primary Antiphospholipid Syndrome Using Gene Microarray Analysis

      Frampton, G.; Murphy, John; Hamid, Colleen G. (University of Wolverhampton, 2007-10)
      Anti-Beta2-Glycoprotein I antibodies (anti-2GPI) are strongly associated with thrombosis in patients with primary antiphospholipid syndrome (PAPS). Anti-2GPI activate endothelial cells (EC) resulting in a pro-thrombotic and pro-inflammatory phenotype. In order to characterise EC gene regulation in response to anti-2GPI, early global gene expression was assessed in human umbilical vein endothelial cells (HUVEC) in response to affinity purified anti-2GPI. Sera were collected from patients with PAPS and IgG was purified using HiTrap Protein G Sepharose columns. Polyclonal anti-2GPI were prepared by passing patient IgG through NHS activated sepharose coupled to human 2GPI. Anti-2GPI preparations were characterized by confirming their 2GPI co-factor dependence, binding to 2GPI and ability to induce leukocyte adhesion molecule expression and IL-8 production in vitro. Two microarray experiments tested differential global gene expression in 6 individual HUVEC donors in response to 5 different PAPS polyclonal anti-2GPI (50 mg/ml) compared to 5 normal control IgG (50 mg/ml) after 4 hours incubation . Total HUVEC RNA was extracted and cRNA was prepared and hybridised to Affymetrix HG-133A (Exp.1) and HG-133A_2 (Exp.2) gene chips. Data were analyzed using a combination of the MAS 5.0 (Affymetrix) and GeneSpring (Agilent) software programmes. Significant change in gene expression was defined as greater than two fold increase or decrease in expression (p<0.05). Novel genes not previously associated with PAPS were induced including chemokines CCL20, CXCL3, CX3CL1, CXCL5, CXCL2 and CXCL1, the receptors Tenascin C, OLR1, IL-18 receptor 1 and growth factors, CSF2, CSF3, IL-6, IL1 and FGF18. Downregulated genes were transcription factors/signaling molecules including ID2. Microarray results were confirmed for selected genes (CSF3, CX3CL1, FGF18, ID2, SOD2, Tenascin C) using quantitative real-time RT-PCR analysis. This study revealed a complex anti-2GPI-regulated gene expression profile in HUVEC in vitro. The novel chemokines and pro-inflammatory cytokines identified in this study may contribute to the vasculopathy associated with PAPS.
    • Signaling pathway of ginsenoside-Rg1 leading to nitric oxide production in endothelial cells.

      Leung, Kar Wah; Cheng, Yuen-Kit; Mak, Nai Ki; Chan, Kelvin C.; Fan, T. P. David; Wong, Ricky N. S. (Elsevier, 2006)
      We here provide definitive evidence that ginsenoside-Rg1, the pharmacologically active component of ginseng, is a functional ligand of the glucocorticoid receptor (GR) as determined by fluorescence polarization assay. Rg1 increased the phosphorylation of GR, phosphatidylinositol-3 kinase (PI3K), Akt/PKB and endothelial nitric oxide synthase (eNOS) leading to increase nitric oxide (NO) production in human umbilical vein endothelial cell. Rg1-induced eNOS phosphorylation and NO production were significantly reduced by RU486, LY294,002, or SH-6. Also, knockdown of GR completely eliminated the Rg1-induced NO production. This study revealed that Rg1 can indeed serve as an agonist ligand for GR and the activated GR can induce rapid NO production from eNOS via the non-transcriptional PI3K/Akt pathway.